The origin of replication in the dihydrofolate reductase (DHFR) domain was the first mammalian origin to be identified and has been characterized by virtually every available replicon mapping technique. This complex origin consists of a hierarchy of at least 25 inefficient initiation sites distributed throughout the 55-kb spacer between the DHFR and 2BE2121 genes. Quantitative high-resolution assays that measure the frequency of initiation at different sites define a central "dead" zone that effectively divides the origin in half, with the center of each half (termed ori-beta and ori-gamma) representing the most active initiation sites in the spacer. Remarkably, deletions of the most active sites, or even the entire 45-kb core of the spacer in which >90% of initiations occur, have no overt negative effects on initiation in the remainder of the spacer in each case nor on the time of replication of the locus as a whole. Thus, if ori-beta and ori-gamma correspond to classic genetic replicators, they must control initiation only in their immediate environments. Furthermore, replicators must be distributed at frequent intervals throughout the spacer that can be activated when neighboring replicators are deleted. Although master replicators do not appear to reside within the origin itself, removal of either the 5' or the 3' end of the DHFR gene results in complete loss of early-firing origin activity in the intergenic spacer. The former result suggests that transcription may be required to somehow activate the intergenic region in early S-phase, while the latter result suggests that high-level transcription through a region inactivates it as a template for initiation of replication. To accommodate all of these observations, we propose a model in which degenerate replicators with different intrinsic affinities for initiation proteins are distributed at 1-2-kb intervals throughout higher eukaryotic genomes, but their activities are regulated by contextual factors such as local transcription, inhibitory boundary elements, attachment to the nuclear matrix, chromatin architecture, etc. Specific aims of this proposal are as follows. Aim 1. To test the proposal that potential replicators are distributed throughout mammalian genomes (including the bodies of active genes) and, given a permissive environment, can attract initiation proteins to their immediate neighborhoods. Aim 2. To test the proposal that the activity of ori-beta can be modulated by changing its position vis-a-vis the end of the gene and the centered "dead" zone. Aim 3. To test the proposal that the shape of the initiation frequency curves in the spacer are defined by read-through DHFR transcription and by the novel transcript in the central "dead"zone. Aim 4. To test whether transcription through the DHFR gene, as opposed to the integrity of the DHFR promoter, is required to activate the intergenic origin, and to identify those elements in the promoter that are required to effect initiation. Aim 5.To determine the distribution of relevant initiation proteins in the DHFR origin, and gain insight into why this origin is so inefficient.